The actions of many neurotransmitters and peptide hormones proceed through receptor-mediated pathways that activate the CAMP-dependent protein kinase (PKA). The diverse biochemical effects of different neurotransmitters or hormones that stimulate CAMP production have been proposed to occur through the activation of particular PKA subtypes at defined subcellular locations. Site-specific localization of the type II PKA is maintained through protein-protein interactions between the regulatory subunit (RII) and anchoring proteins. We propose that RII-anchoring proteins dictate the localization of type II PKA an thereby position the kinase close to substrate proteins. The overall goals of this project are to determine the structures that promote RII-anchoring protein interaction and to elucidate the roles of selected anchoring proteins in dictating the cellular distribution and function of PKA. The specific aims are to: 1) determine the structural requirements for RII-anchoring protein interaction by mutagenesis of the regulatory subunit. Complementary studies will characterize the RII- binding domain of MAP 2 and Ht 21, a thyroid RII-anchoring protein identified in our laboratory; 2) characterize novel RII-anchoring protein CDNAS isolated by a RII-binding assay from a thyroid lambda gt11 library; and 3) test the hypothesis that the subcellular localization of type II RII PKA is determined by the location of specific anchoring proteins in vivo by overexpressing anchoring proteins and anchoring protein fragments in the thyroid cell line FRTL 5.